Our overall goal is to determine how the initiation of coliphage lambda DNA replication is interlocked and coupled to transcriptional controls. We hypothesize that initiation of replication involves interaction of initiator proteins at the origin of replication (ori) site resulting in transcription of a small RNA molecule which primes DNA synthesis. Previous studies have shown that transcription and replication are coupled in lambda. Several E. coli (dnaB and dnaG) and lambda (ori-, O-, and P-) mutations inhibit the initiation of lambda DNA replication and the synthesis of a unique 4S RNA, termed oop, transcribed from within the ori region. We will determine whether the initiation of lambda replication required the prior transcription of oop RNA and if oop primes DNA synthesis. Techniques of multistep liquid DNA-RNA hybridization will be used (a) to study the control of initiation of oop synthesis (b) to explain the activity of the replication initiator proteins, and (c) to distinguish the requirement of factors for initiation versus elongation. We will map, isolate and fingerprint minor RNAs which are transcribed from the 1-strand Q-R-A-J and r-strand int-cI regions and determine if these RNAs are involved in synthesis of discontinuously replicated DNA. In xis- phage these two regions cannot be transcribed from pL or pR and must be replicated discontinuously. The initiation sites of oop-stimulated in vitro DNA synthesis will be mapped by electron microscopy. We will determine if oop is covalently joined to in vitro and in vivo initiated lambda DNA synthesis. These studies can contribute to an understanding of the loss of normal control over the initiation of new rounds of replication which occurs in proliferative cell diseases.